Pathogenesis of Keratin-Containing Inclusions in Liver Disease

肝脏疾病中含角蛋白包涵体的发病机制

• 批准号: 7905576
• 负责人: Bishr Omary
• 金额: $ 10万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-05 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7905576
• 关键词: Address; Amino Acids; Apoptosis; Area; Cell Culture Techniques; Coupled; Cytoprotection; Cytoskeletal Proteins; Disease Association; Disease Progression; Epithelium; Functional disorder; Glutamine; Goals; Hepatitis; Hepatocyte; Human; In Vitro; Inclusion Bodies; Injury; Injury to Liver; Intermediate Filament Proteins; Intermediate Filaments; K-18 conjugate; Keratin; Knockout Mice; Light; Liver; Liver diseases; Lysine; Mallory Body; Mechanics; Memory; Modeling; Molecular; Molecular Chaperones; Mus; Mutation; Neurologic; Neurons; Organ; Pathogenesis; Pharmaceutical Preparations; Phosphorylation; Predisposition; Proteins; Recovery; Regulation; Role; Site; Staging; Stress; Testing; Tissue Polypeptide Specific Antigen; Transgenic Animals; Transgenic Mice; Transglutaminases; Up-Regulation; amino group; base; carboxyl group; clinically relevant; crosslink; genetic variant; glycosylation; human disease; in vivo; in vivo Model; mutant; neuromuscular; polypeptide; problem drinker; promoter; protein function; transglutaminase 2

DESCRIPTION (provided by applicant): The overall goal of our studies is to understand the regulation, function, and disease association of the keratin intermediate filament (IF) cytoskeletal proteins in digestive organs. Keratins 8 and 18 (K8/K18) are the IFs of hepatocytes and their major function is cytoprotection from mechanical and nonmechanical stresses such as apoptosis. This function is consistent with the finding that specific K8 and K18 genetic variants predispose to liver disease progression. K8 and K18 are also the major constituents of Mallory body (MB) inclusions that are found in association with some liver diseases. Several transgenic animal studies demonstrated that K8 and a K8-greater-than-K18 protein ratio, coupled with a drug insult are all essential in order for MBs to form. MBs can be rapidly re-induced once initially formed but the mechanism of such rapid predisposition to reformation is unknown. Our proposal includes 4 aims to examine the pathogenesis of MBs. The first three aims test the hypothesis that keratin transamidation at specific amino acids is essential for MB formation. The 4th aim tests the hypothesis that chaperone function is altered during MB (re)formation. The proposed studies utilize cell culture and mouse MB models to help understand their pathogenesis and, ultimately, their importance in human disease. The 4 aims are: (i) Study MB formation in transglutaminase-2 (TG2) null and control mice using established in vivo models. This aim is based on our preliminary findings that TG2-null mice have a markedly blunted ability to form MBs. (ii) Identify keratin transamidation sites and their crosslinked partners in vivo/vitro and study their role in crosslinking and inclusion body formation in cell culture. This is based on the findings that K8/K18 are excellent TG2 substrates (K8>K18) in vitro, (iii) Generate transgenic mice that express transamidation-mutant keratins and test the effect of the mutations on MB formation and susceptibility to liver injury, (iv) Test the effect of MB-inducing injury on chaperone function and the potential role of chaperones as "memory proteins" in association with MB re-accumulation. This is based on our recent findings that MB formation correlates with chaperone dysfunction. Our proposed studies are likely to generate important new biologic and clinically relevant information regarding the pathogenesis of MBs. We anticipate that completion of this project will shed light on the specific keratin amino acids that allow MB formation and address if MBs are bystanders, protectors or promoters of injury. Our findings should also shed light on whether chaperone dysfunction can provide 'molecular memory" and as such contribute to rapid MB reformation. Our proposal represents a direct approach to fully understand the pathogenesis and significance of MBs that were first described in 1911 by Dr. Frank Mallory, and may also impact on other inclusions that are found in association with several other neurological and neuromuscular human diseases whose pathogenesis is unknown.

描述（由申请人提供）：我们研究的总体目标是了解消化器官中角蛋白中间丝（IF）细胞骨架蛋白的调控、功能和疾病相关性。角蛋白8和18 （K8/K18）是肝细胞的干扰素，其主要功能是保护细胞免受机械和非机械应激（如凋亡）的影响。这一功能与特定的K8和K18基因变异易导致肝脏疾病进展的发现是一致的。K8和K18也是与某些肝脏疾病相关的Mallory小体（MB）内含物的主要成分。几项转基因动物研究表明，为了形成MBs， K8和K8-大于k18的蛋白质比例，加上药物损伤都是必不可少的。MBs一旦最初形成，可以迅速重新诱导，但这种快速重组倾向的机制尚不清楚。我们的提案包括4个目的来研究MBs的发病机制。前三个目的是测试特定氨基酸的角蛋白转氨化对MB形成至关重要的假设。第4个目的是验证在MB （re）形成过程中伴侣蛋白功能发生改变的假设。拟议的研究利用细胞培养和小鼠MB模型来帮助了解其发病机制，并最终了解其在人类疾病中的重要性。4个目标是：(i)使用已建立的体内模型研究转谷氨酰胺酶-2 （TG2）缺失和对照小鼠中MB的形成。这一目标是基于我们的初步发现，tg2缺失小鼠形成MBs的能力明显减弱。（ii）在体内/体外鉴定角蛋白转酰胺位点及其交联伙伴，并研究其在细胞培养中交联和包涵体形成中的作用。这是基于K8/K18在体外是优秀的TG2底物（K8>K18）的发现，（iii）产生表达转酰胺突变角蛋白的转基因小鼠，并测试突变对MB形成和肝损伤易感性的影响，（iv）测试MB诱导损伤对伴侣蛋白功能的影响以及伴侣蛋白作为“记忆蛋白”与MB再积累相关的潜在作用。这是基于我们最近的发现，MB的形成与伴侣功能障碍有关。我们提出的研究可能会产生关于MBs发病机制的重要的新的生物学和临床相关信息。我们预计，该项目的完成将揭示特定的角蛋白氨基酸，允许MB形成和解决MBs是旁观者，保护或促进损伤。我们的研究结果还揭示了伴侣功能障碍是否可以提供“分子记忆”，从而有助于MB的快速重组。我们的研究为充分理解Frank Mallory博士于1911年首次描述的MBs的发病机制和意义提供了一种直接的方法，也可能影响与其他几种发病机制未知的神经和神经肌肉疾病相关的其他包涵体。